SAR Analysis of Antagonists of the MOR Receptor using an Image-Based Assay - Set 3

Drug addiction is a disease that involves the G-protein coupled receptors in the central nervous system resulting in compulsive or abnormal behavior. Recent studies have shown that opioid receptors play a role in regulating other receptors that interact with drug and other substance abuse. The opioid receptors are composed of multiple of multiple receptor subtypes whose contribution the addictive behaviors are not fully delineated. ..more

Drug addiction is a disease that involves the G-protein coupled receptors in the central nervous system resulting in compulsive or abnormal behavior. Recent studies have shown that opioid receptors play a role in regulating other receptors that interact with drug and other substance abuse. The opioid receptors are composed of multiple of multiple receptor subtypes whose contribution the addictive behaviors are not fully delineated.

The aim of this assay is to identify small molecule antagonists of the human mu opioid receptor (MOR). This dose response assay is developed and performed to confirm hits originally identified in "uHTS identification of small molecule agonists of the kappa opioid receptor via a luminescent beta-arrestin assay" (AID 1777) and to study the structure-activity relationship on analogs of the confirmed hits. Compounds are either acquired from commercial sources or synthesized internally.

This image-based MOR agonist assay utilizes a cell line permanently expressing a beta-arrestin GFP biosensor and the mu opioid receptor (MOR). Upon agonist-mediated GPCR activation, the arrestin-GFP redistributes from the cytosolic compartment to the plasma membrane and further to coated pits / endosomal vesicles, which can be quantified as increased local aggregation of the GFP-arrestins.

Assay Procedure:1) 45ul of cell suspension (200,000 cells/ml in culture medium) was dispensed in each well of the assay plates using a Wellmate bulk dispenser. 2) Plates are incubated overnight or approximately 20 hours at 37 degree C and 5% CO2.3) Serum is removed by media aspiration and replaced with 45ul serum-free MEM prior to addition of compounds. 4) Compound addition was done on the ECHO550 Liquid Handler. The "dose response protocol" was used to dispense corresponding volumes of each 10mM compound on the assay plate.a. Compounds were added to columns 3 to 22. Final concentration ranged from 62nM to 32uM (ten doses), in duplicate.b. Naloxone was added to column 1 and 24. Naloxone final concentration was 1uM. d. DMSO was back-filled to each well to achieve a 0.31% final concentration. DMSO final concentration was 0.31%.5) Plates were incubated for 30 minutes at 37 degrees C and 5% CO2. 6) Damgo was added to the entire plate, except column 23. Damgo final concentration was 5uM. Plates were then incubated for another 45 minutes. 7) Media was aspirated leaving 20ul liquid in each well using a Titertek plate washer. 8) 40ul of fixative working solution was added to each well using a Wellmate bulk dispenser (Matrix) for a final concentration of 4% PFA.9) Plates were incubated for 40 minutes at room temperature.10) Fixative was aspirated and plates were washed twice with 50ul PBS leaving 20ul liquid in each well using a Titertek plate washer.11) 40ul of DAPI working solution was added using a Wellmate bulk dispenser for a final DAPI concentration of 100ng/ml. Aluminum plate sealers were applied to each plate.

Compounds with an IC50 < 10 uM are defined as actives in the dose response confirmation.

To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the assay is described below.

Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the assay is as follows: 1) First tier (0-40 range) is reserved for primary screening data and is not applicable to this assay

2) Second tier (41-80 range) is reserved for dose-response confirmation data and is not applicable to this assay

3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues.

a. Inactive compounds of the confirmatory stage are assigned a score value equal 81. b. For the remaining compounds the score is linearly correlated with a compound's inhibitory potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.c. Summary equation that takes into account the items discussed above is Score = 82 + 3*(pIC50 - 3), where pIC50 is a negative log(10) of the IC50 value expressed in mole/L concentration units. This equation results in the Score values above 85 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.

This qualifier is to be used with the next TID, IC50_Mean. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value

String

2

IC50_Mean*

IC50 value determined using sigmoidal dose response equation

Float

μM

3

IC50_Qualifier_1

This qualifier is to be used with the next TID, IC50_1. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value

String

4

IC50_1

IC50 value determined using sigmoidal dose response equation

Float

μM

5

Std.Err(IC50)_1

Standard Error of IC50 value

Float

μM

6

nH_1

Hill coefficient determined using sigmoidal dose response equation

Float

7

Excluded_Points_first_point

Flags to indicate which of the first dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

8

% Activity at 32 uM_first_point (32μM**)

% Inhibition at a given concentration

Float

%

9

% Activity at 16 uM_first_point (16μM**)

% Inhibition at a given concentration

Float

%

10

% Activity at 8 uM_first_point (8μM**)

% Inhibition at a given concentration

Float

%

11

% Activity at 4 uM_first_point (4μM**)

% Inhibition at a given concentration

Float

%

12

% Activity at 2 uM_first_point (2μM**)

% Inhibition at a given concentration

Float

%

13

% Activity at 1 uM_first_point (1μM**)

% Inhibition at a given concentration

Float

%

14

% Activity at 0.5 uM_first_point (0.5μM**)

% Inhibition at a given concentration

Float

%

15

% Activity at 0.25 uM_first_point (0.25μM**)

% Inhibition at a given concentration

Float

%

16

% Activity at 0.125 uM_first_point (0.125μM**)

% Inhibition at a given concentration

Float

%

17

% Activity at 0.0625 uM_first_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

18

Excluded_Points_second_point

Flags to indicate which of the second dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

19

% Activity at 32 uM_second_point (32μM**)

% Inhibition at a given concentration

Float

%

20

% Activity at 16 uM_second_point (16μM**)

% Inhibition at a given concentration

Float

%

21

% Activity at 8 uM_second_point (8μM**)

% Inhibition at a given concentration

Float

%

22

% Activity at 4 uM_second_point (4μM**)

% Inhibition at a given concentration

Float

%

23

% Activity at 2 uM_second_point (2μM**)

% Inhibition at a given concentration

Float

%

24

% Activity at 1 uM_second_point (1μM**)

% Inhibition at a given concentration

Float

%

25

% Activity at 0.5 uM_second_point (0.5μM**)

% Inhibition at a given concentration

Float

%

26

% Activity at 0.25 uM_second_point (0.25μM**)

% Inhibition at a given concentration

Float

%

27

% Activity at 0.125 uM_second_point (0.125μM**)

% Inhibition at a given concentration

Float

%

28

% Activity at 0.0625 uM_second_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

29

IC50_Qualifier_2

This qualifier is to be used with the next TID, IC50_2. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value

String

30

IC50_2

IC50 value determined using sigmoidal dose response equation

Float

μM

31

Std.Err(IC50)_2

Standard Error of IC50 value

Float

μM

32

nH_2

Hill coefficient determined using sigmoidal dose response equation

Float

33

Excluded_Points_third_point

Flags to indicate which of the third dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

34

% Activity at 32 uM_third_point (32μM**)

% Inhibition at a given concentration

Float

%

35

% Activity at 16 uM_third_point (16μM**)

% Inhibition at a given concentration

Float

%

36

% Activity at 8 uM_third_point (8μM**)

% Inhibition at a given concentration

Float

%

37

% Activity at 4 uM_third_point (4μM**)

% Inhibition at a given concentration

Float

%

38

% Activity at 2 uM_third_point (2μM**)

% Inhibition at a given concentration

Float

%

39

% Activity at 1 uM_third_point (1μM**)

% Inhibition at a given concentration

Float

%

40

% Activity at 0.5 uM_third_point (0.5μM**)

% Inhibition at a given concentration

Float

%

41

% Activity at 0.25 uM_third_point (0.25μM**)

% Inhibition at a given concentration

Float

%

42

% Activity at 0.125 uM_third_point (0.125μM**)

% Inhibition at a given concentration

Float

%

43

% Activity at 0.0625 uM_third_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

44

Excluded_Points_fourth_point

Flags to indicate which of the fourth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

45

% Activity at 32 uM_fourth_point (32μM**)

% Inhibition at a given concentration

Float

%

46

% Activity at 16 uM_fourth_point (16μM**)

% Inhibition at a given concentration

Float

%

47

% Activity at 8 uM_fourth_point (8μM**)

% Inhibition at a given concentration

Float

%

48

% Activity at 4 uM_fourth_point (4μM**)

% Inhibition at a given concentration

Float

%

49

% Activity at 2 uM_fourth_point (2μM**)

% Inhibition at a given concentration

Float

%

50

% Activity at 1 uM_fourth_point (1μM**)

% Inhibition at a given concentration

Float

%

51

% Activity at 0.5 uM_fourth_point (0.5μM**)

% Inhibition at a given concentration

Float

%

52

% Activity at 0.25 uM_fourth_point (0.25μM**)

% Inhibition at a given concentration

Float

%

53

% Activity at 0.125 uM_fourth_point (0.125μM**)

% Inhibition at a given concentration

Float

%

54

% Activity at 0.0625 uM_fourth_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

55

IC50_Qualifier_3

This qualifier is to be used with the next TID, IC50_3. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value

String

56

IC50_3

IC50 value determined using sigmoidal dose response equation

Float

μM

57

Std.Err(IC50)_3

Standard Error of IC50 value

Float

μM

58

nH_3

Hill coefficient determined using sigmoidal dose response equation

Float

59

Excluded_Points_fifth_point

Flags to indicate which of the fifth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

60

% Activity at 32 uM_fifth_point (32μM**)

% Inhibition at a given concentration

Float

%

61

% Activity at 16 uM_fifth_point (16μM**)

% Inhibition at a given concentration

Float

%

62

% Activity at 8 uM_fifth_point (8μM**)

% Inhibition at a given concentration

Float

%

63

% Activity at 4 uM_fifth_point (4μM**)

% Inhibition at a given concentration

Float

%

64

% Activity at 2 uM_fifth_point (2μM**)

% Inhibition at a given concentration

Float

%

65

% Activity at 1 uM_fifth_point (1μM**)

% Inhibition at a given concentration

Float

%

66

% Activity at 0.5 uM_fifth_point (0.5μM**)

% Inhibition at a given concentration

Float

%

67

% Activity at 0.25 uM_fifth_point (0.25μM**)

% Inhibition at a given concentration

Float

%

68

% Activity at 0.125 uM_fifth_point (0.125μM**)

% Inhibition at a given concentration

Float

%

69

% Activity at 0.0625 uM_fifth_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

70

Excluded_Points_sixth_point

Flags to indicate which of the sixth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

71

% Activity at 32 uM_sixth_point (32μM**)

% Inhibition at a given concentration

Float

%

72

% Activity at 16 uM_sixth_point (16μM**)

% Inhibition at a given concentration

Float

%

73

% Activity at 8 uM_sixth_point (8μM**)

% Inhibition at a given concentration

Float

%

74

% Activity at 4 uM_sixth_point (4μM**)

% Inhibition at a given concentration

Float

%

75

% Activity at 2 uM_sixth_point (2μM**)

% Inhibition at a given concentration

Float

%

76

% Activity at 1 uM_sixth_point (1μM**)

% Inhibition at a given concentration

Float

%

77

% Activity at 0.5 uM_sixth_point (0.5μM**)

% Inhibition at a given concentration

Float

%

78

% Activity at 0.25 uM_sixth_point (0.25μM**)

% Inhibition at a given concentration

Float

%

79

% Activity at 0.125 uM_sixth_point (0.125μM**)

% Inhibition at a given concentration

Float

%

80

% Activity at 0.0625 uM_sixth_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

81

IC50_Qualifier_4

This qualifier is to be used with the next TID, IC50_4. If qualifier is "=", the IC50 result equals the value in that column. If the qualifier is ">", the IC50 result is greater than that value. If the qualifier is "<", the IC50 result is smaller than that value

String

82

IC50_4

IC50 value determined using sigmoidal dose response equation

Float

μM

83

Std.Err(IC50)_4

Standard Error of IC50 value

Float

μM

84

nH_4

Hill coefficient determined using sigmoidal dose response equation

Float

85

Excluded_Points_seventh_point

Flags to indicate which of the seventh dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.

String

86

% Activity at 32 uM_seventh_point (32μM**)

% Inhibition at a given concentration

Float

%

87

% Activity at 16 uM_seventh_point (16μM**)

% Inhibition at a given concentration

Float

%

88

% Activity at 8 uM_seventh_point (8μM**)

% Inhibition at a given concentration

Float

%

89

% Activity at 4 uM_seventh_point (4μM**)

% Inhibition at a given concentration

Float

%

90

% Activity at 2 uM_seventh_point (2μM**)

% Inhibition at a given concentration

Float

%

91

% Activity at 1 uM_seventh_point (1μM**)

% Inhibition at a given concentration

Float

%

92

% Activity at 0.5 uM_seventh_point (0.5μM**)

% Inhibition at a given concentration

Float

%

93

% Activity at 0.25 uM_seventh_point (0.25μM**)

% Inhibition at a given concentration

Float

%

94

% Activity at 0.125 uM_seventh_point (0.125μM**)

% Inhibition at a given concentration

Float

%

95

% Activity at 0.0625 uM_seventh_point (0.0625μM**)

% Inhibition at a given concentration

Float

%

96

Excluded_Points_eighth_point

Flags to indicate which of the eighth dose-response points were excluded from analysis. (1) means the titration point was excluded and (0) means the point was not excluded.